Fig. 2

Strategy for evaluating hybrid immunoprecipitation HR-PRM-MS method variability. The variability in the IP-HR-PRM-MS method targeting selected LAMP2 peptides was evaluated by employing two methodological pathways, Workflow 1 and Workflow 2. Workflow 1 targeted the overall variability of the method whereas Workflow 2 was designed to isolate the variability in the LC-HR-PRM-MS analysis. Using a single QC pool of human CSF LAMP2 was immunoprecipitated on three separate occasions, Batch 1–3, which contained 8 + 8 replicate samples each (eight for each workflow). This enabled determination of intra- and inter-day variation. After the immunoprecipitation, isotope labeled LAMP2 peptides corresponding to the peptides previously identified using nano-LC tandem MS were added as well as full length BSA and an isotope labeled BSA peptide. The samples were then digested by trypsin. In Workflow 1 each sample was processed individually from beginning to end, thus reflecting the overall methodological variability. In Workflow 2 the eight replicate samples in each batch were pooled after trypsination. Thus, Workflow 2 was equivalent to eight injections of the same sample, for the respective batch, in the micro-LC HR-PRM-MS analysis. Although the different batches were prepared on different occasions they were all analyzed on a single occasion to minimize the influence of altering instrumental performance